Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane
February 8th, 2020
Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.
Clear cell renal cell carcinoma or CCRCC, is the most common subtype of kidney cancer. Localize CCRCC has favorable outcome with surgery. However, one third of CCRCC patients will develop distant metastasis to the lung.
Which leads to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage. As the molecular mechanism of metastasis remains allusive.
The purpose of this protocol is to introduce and compare the two animal models recently established for metastatic CCRCC. The first model is the mouse of the topic implantation. In this mouse model, heterogeneous CCRCC is implanted to the renal capsule of the mouse.
Tumor growth and the metastasis are asset after 10 weeks. The second model is the chicken chorioallantoic membrane model, or the CAM model. In this chicken model the heterogeneous CCRCC is implanted in the egg.
Tumor growth and circulating tumor cells are asset after 10 days of implantation before the embryo hatches. Lung metastasis is asset after 24 days of the implantation. Which is two weeks after hatching.
These two novel models will be useful to further study the molecular mechanism behind the metastasis in CCRCC. Please note that the CAM procedure is not featured in this video. The detailed protocol for the CAM model is described and filmed in another Trove article by Sharrow et al.
After anesthetizing a the mouse, disinfect the back of the mouse entirely with povidone-iodine for three times. Follow by 70%ethnol once, and wipe it dry with sterile cotton swabs. Then apply three sterile medical dressing sequentially covering the whole back in order to create a surgical field as well as to immobilize the mouse.
Before the operation, the operators should disinfect their fingers with povidone-iodine. Place the mouse in the prone position. And, use fingers to locate the left kidney just under the left flank.
Use a pair of blunt forceps and scissors to cut open the skin and muscle layer under the specified location. Use sterile cotton swabs and forceps to partially exteriorize the left kidney out of the abdomen. Load either an insulin syringe or a customized Hamilton syringe with the tumor cell suspension prepared separately.
Inject 20 microliters of cell suspension under the kidney capsule. Slowly pull out needle in order to solidify the extra cellular matrix solution. As well as to prevent hemorrhage or tumor cell leakage.
And then, use a sterile cotton swab to push the kidney back into the abdomen. Use suture to stitch the muscle layer. Disinfect the skin with povidone-iodine.
And, close the skin with wound auto-clips. Orthotopic laying planted tumor cells successfully grew on mice kidney as confirmed by BLI. Although there was no difference in the primary growth, only the heterogeneous metastatic tumor had robust metastasis to the lung.
The metastasis was also confirmed by immunohistochemistry. Similarly, the circulating tumor cell counts were higher in the mice bearing metastatic tumor than those bearing non-metastatic tumor. In concordance with the mouse model the CAM model also showed no growth difference between metastatic and non-metastatic tumors.
On the other hand, the number of circulating tumor cells was significantly higher in the eggs with metastatic tumors. Histologically detectable metastasis was observed in the lungs of hatched chicken. In conclusion, this protocol illustrated two novel animal models for metastatic CCRCC.
Both the mouse and the chicken models faithfully recapitulated the lung metastasis observed in the clinic. These two pre-clinical models will be useful to expand the understanding of the molecular mechanism behind the metastasis. As well as to discover novel treatments for metastatic CCRCC.
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Overview
This article presents two novel animal models for metastatic clear cell renal cell carcinoma (CCRCC), addressing the lack of comprehensive models for preclinical investigation. The models include an orthotopically implanted mouse model and a chicken chorioallantoic membrane model, both demonstrating lung metastasis similar to clinical cases.
Key Study Components
Area of Science
- Oncology
- Animal Models
- Metastasis Research
Background
- Clear cell renal cell carcinoma (CCRCC) is the most common subtype of kidney cancer.
- Localized CCRCC has a favorable outcome with surgery.
- One third of CCRCC patients develop distant metastasis, particularly to the lung.
- There is currently no effective therapy for this advanced stage of the disease.
Purpose of Study
- To develop and validate new animal models for studying CCRCC metastasis.
- To facilitate preclinical investigations into the disease mechanisms.
- To improve understanding of lung metastasis in CCRCC.
Methods Used
- Orthotopic implantation of tumor cells in mice.
- Utilization of the chicken chorioallantoic membrane model.
- Observation of lung metastasis development in both models.
- Comparison of model outcomes with clinical cases.
Main Results
- Both animal models successfully demonstrated lung metastasis.
- The models closely resemble clinical cases of CCRCC.
- These models provide a platform for further research into therapeutic strategies.
- Insights into the molecular mechanisms of metastasis were gained.
Conclusions
- The newly developed models are valuable for preclinical studies of CCRCC.
- They enhance the understanding of metastatic processes in kidney cancer.
- Future research can leverage these models to explore potential therapies.
Frequently Asked Questions
Executive Industry Relevance
Metastatic clear cell renal cell carcinoma (ccRCC) remains a high unmet need due to lack of effective therapies and poor understanding of metastatic mechanisms. The orthotopic mouse and chicken chorioallantoic membrane (CAM) models described provide complementary preclinical systems to study lung metastasis, enabling target validation and mechanistic de-risking in discovery pipelines. These models support predictive confidence in therapeutic development by recapitulating clinical metastasis patterns in immunocompetent and cost-efficient platforms.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of VHL loss-driven tumorigenicity and metastatic potential in heterogeneous ccRCC populations.
- Operational Value: Orthotopic implantation in immunocompetent mice preserves tumor-stroma interactions critical for target validation.
- Scientific Value: Facilitates functional validation of metastasis-associated genes and pathways through in vivo lung colonization assays.
Screening & Assay Development
- Scientific Value: Generates quantifiable circulating tumor cell (CTC) counts as a pharmacodynamic readout for compound screening.
- Operational Value: CAM model offers rapid, cost-efficient tumor growth (10 days) for high-throughput assay standardization.
- Scientific Value: Supports intravasation assessment via CTC detection in both mouse and CAM systems, enabling early-stage compound evaluation.
Translational & Preclinical Research
- Scientific Value: Models recapitulate clinical lung metastasis, providing disease-relevant systems for biomarker discovery and validation.
- Operational Value: Extended CAM model in hatched chickens (24 days) enables micrometastasis detection by immunohistochemistry for longitudinal studies.
- Scientific Value: Supports patient-derived xenograft (PDX) establishment from heterogeneous tumor populations for preclinical continuity.
Pipeline & Workflow Integration
The models integrate across discovery stages: orthotopic mouse models support hypothesis testing and pathway validation in early discovery, while CAM enables rapid assay development and screening readiness. Both systems generate quantitative outputs such as tumor burden, CTC levels, and metastatic lesion quantification, informing go/no-go decisions. The models serve as reusable platforms for target de-risking and translational biomarker alignment, reducing biological risk in lead identification.
- Discovery Biology: Validates therapeutic targets through assessment of tumor growth and lung metastasis in immunocompetent hosts.
- Screening: Provides standardized, reproducible systems for CTC-based pharmacodynamic monitoring in compound screening campaigns.
- Analytics: Enables quantitative comparison of metastatic burden via bioluminescence imaging (BLI) and immunohistochemistry (IHC) readouts.
- Translational Research: Connects discovery to preclinical validation through PDX-compatible heterogeneous tumor models.
- Enterprise Reuse: Establishes a scalable, cross-platform capability for metastasis model generation across multiple therapeutic areas.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity in metastasis research by providing clinically relevant lung metastasis models.
- Operational Value: CAM model reduces time, labor, and cost versus traditional mouse models while supporting robust tumor formation and intravasation.
- Strategic Value: Improves portfolio triage by enabling early assessment of metastatic potential and therapeutic efficacy.
- Portfolio Impact: Supports risk-adjusted advancement decisions through quantitative metastasis endpoints in preclinical validation.
Implementation Considerations
- Requires expertise in orthotopic surgical techniques and tumor cell preparation for mouse model implantation.
- Dependent on sterile surgical infrastructure and anesthesia protocols for murine procedures.
- Necessitates access to embryonated eggs and incubation facilities for CAM model execution.
- Requires standardization of tumor cell suspension preparation and injection volumes (20 µL) across models.
- Relies on histopathological validation via BLI and IHC for metastasis detection, necessitating imaging and pathology resources.
